Project C ? Structural Studies of Microtubule Dynamics and Interactions in Mitosis Eva Nogales PROJECT SUMMARY/ABSTRACT Microtubules (MTs) are cytoskeletal polymers of ??-tubulin that play essential roles in all eukaryotic cells. MTs form the mitotic spindle, a dynamic suprastructure that separates duplicated chromosomes equally between daughter cells during cell division. Central to this function is the property of dynamic instability by which MTs switch between growing and shrinking phases powered by GTP hydrolysis. Anticancer drugs that bind to tubulin stop cell division by interfering with MT dynamics. On the other hand, many MT cellular partners modulate dynamic instability or utilize it to carry out specific functions. Among the first are +TIPs, proteins that track MT growing ends and affect their dynamic behavior. Among the second are kinetochore complexes ?protein assemblies that connect chromosomes to spindle MTs? that are able to harness the energy of MT depolymerization to move chromosome during cell division. We are interested in deciphering the molecular mechanisms by which MTs undergo dynamic instability, how this property is regulated by cellular factors and antimitoti agents, and how MT dynamics are used by kinetochore components for chromosome movement. Towards these goals we are using cryo-electron microscopy (cryo-EM) to provide unique structural insight into physiologically relevant structures of MTs in different nucleotide and drug states, and visualize the interactions between MTs and a +TIP protein (EB3), and between MTs and large kinetochore complexes. For the biochemically simpler studies we aim for atomic resolution detail concerning changes in tubulin structure that drive MT disassembly or stabilization by EB3 or drugs. Such studies rely on state- of-the-art cryo-EM equipment and on our novel data processing strategy to take advantage of the pseudo- helical symmetry of MTs. These studies will provide an unprecedented pool of mechanistic information concerning MT dynamic instability, its obliteration by important anticancer agents, and one major cellular strategy for its regulation. Our structures will constitute unique material for computational studies aiming at modeling physical properties of MTs or at uncovering new potential sites for drug design to control MT behavior/cell proliferation, and ultimately fight disease. Our ambitious MT-kinetochore interaction studies also rely on state of the art equipment and will involve an innovative bootstrapping structural approach where molecular complexity and experimental difficulty are build up one step at a time, and where the interpretation of each EM structure builds on the previous step in a robust manner. Our overreaching goal is the structural characterization of a complete kinetochore and its regulated interaction with MTs. These studies will shed mechanistic light into mitosis and contribute to the improvement or development of anticancer agents that interfere with the normal or diseased mitotic process.